In this report we describe a cDNA sequence, BS106, identified from Incyte Genomics LifeSeq® Expressed Sequence Tag database. A multi-tissue mRNA expression array, northern blots, and RT-PCR assays demonstrate the expression of BS106 in mammary, salivary and prostate glands, but not in other tissue types. BS106 mRNA was detected in 90% of the breast tissues examined. The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein based on amino acid composition, extensive O-linked glycosylation, and expression profile. BS106 protein was recombinantly expressed in human embryonic kidney 293 cells and the secreted product was purified from the culture media. Monoclonal antibodies were prepared and used for immunohistochemical analysis of early stage breast cancer. BS106 protein was detected in the vast majority of carcinomas (70–100%) and overexpressed in approximately 30% of the 22 specimens analyzed. BS106 protein was not detected in other solid tumor types including bladder carcinoma, colon carcinoma, endometrial carcinoma, gastric carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, ovarian carcinoma, pancreatic and prostatic carcinoma.
The concept of antigen-directed cell fusions to increase the yield of hybridomas was investigated. To facilitate cell-cell contact, antigen conjugated cells were used in cell fusion studies. Specifically, fluorescyl conjugated murine myeloma cells (Sp 2/0-Ag14) incubated with murine immune (anti-fluorescyl) splenocytes formed aggregates containing fluorescent and non-fluorescent cells. Fusion of these populations with polyethylene glycol resulted in a greater number of anti-fluorescyl hybridomas relative to normal fusions under non-antigen directed condition. Ligand binding data indicated that despite the multi-cellular aggregates the hybridomas resulting from chemically mediated fusions produced only one monoclonal Ig.
The ability of rat serum to inactivate endotoxin (LPS) was assessed with the aid of the limulus amebocyte lysate assay. Following the addition of various amounts of endotoxin to normal serum the mixture was incubated for 1 hr at 37°C and the residual endotoxin activity determined. One milliliter of rat serum inactivated between 5 and 10 pg Escherichia coli LPS per hour. Heating serum for 45 min at 56°C resulted in loss of 80-90% of the LPS inhibitor (LPSI) activity. Serum from cobra venom factor (CVF)-treated rats inactivated between 0.5 and 2.5 pg LPS/ml serum. Serum from tolerant rats, even after heating for 45 min at 56"C, inactivates between 10 and 15 pg LPS/ml serum/hr; decomplemented tolerant rat serum neutralizes between 5 and 10 pg LPS/ml serum/hr. Clearly, the tolerant rat has large quantities of LPSI activity, which does not appear to be complement. The inhibitor found in tolerant rat serum is not species specific since it inactivates Salmonella minnesota and Salmonella typhimurium endotoxins to the same degree and in the same amount as E. coli endotoxin, the agent used to induce tolerance. Both heating serum (56°C) and lead acetate reduce LPSI activity. o
The effect of acute hepatotoxin exposure on in vivo and in vitru immune responses were investigated in inbred mice. Splenic anti-SRBC PFC responses were slightly enhanced by carbon tetrachloride or galactosamine administration 5 hr prior to immunization. Whereas splenic anti-SRBC PFC responses were slightly enhanced in euthymic mice exposed to carbon tetrachloride 5 hr prior to immunization, immune responses to the TI antigens, Fl-LPS, Fl-Ficoll, and TNP-LPS, were significantly suppressed. Athymic mice receiving similar hepatotoxin exposure elicited enhanced immune responses to the TI immunogens, thereby suggesting that the activities of B cells and macrophages are enhanced in treated animals and in euthymic mice, T suppressor cells are also activated. By admixture of purified B-and T-cell and macrophage populations from either carbon tetrachloride-treated or control animals, it was demonstrated that hepatotoxin exposure also induces suppressor T cells regulating immune responses to the Tdependent antigen, SRBC, and that macrophages from treated animals are more functional. Further, B-cell responsiveness is enhanced. In addition to these observations, an active factor could be demonstrated in sera from hepatotoxin-treated animals which augments immune responses to SRBC in normal mice and promotes immune responses to this antigen in athymic mice. These findings indicate that the effects of acute hepatotoxin exposure are multifocal, influencing the activity of lymphoid and accessory cells.
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